The present invention relates to the general field of steroid chemistry, particularly to estrone derivatives, and more particularly to B-ring expanded estra-1,3,5(10)-triene compounds. The invention also relates to the field of anti-mitotic, anti-tumor, and anti-angiogenic therapeutics, particularly to the field of therapeutics that function by modulation of the polymerization of tubulin and/or the depolymerization of microtubules.
1 Tubulin polymerization and Microtubule Assembly and Disassembly.
Cell mitosis is a multi-step process that includes chromosome replication and cell division. It is characterized by the intracellular movement and segregation of organelles, including mitotic spindles and the replicated chromosomes. Organelle movement and segregation are dependent upon the polymerization of the heterodimeric cell protein tubulin into structures called microtubules. Successful cell division is therefore dependent upon the proper polymerization of tubulin, and also upon the proper functioning and subsequent disassembly of the resulting microtubules.
Tubulin and microtubules are sensitive to a variety of antimitotic drugs. For example, colchicine and nocadazole are anti-mitotic drugs that bind tubulin and inhibit tubulin polymerization, preventing microtubule formation. As such they have anti-tumor activity. In contrast, the anti-cancer drug TAXOL(trademark) binds to and stabilizes the microtubules, inhibiting depolymerization and thereby interfering with the later stages of mitosis. Thus, compounds that inhibit either the polymerization or depolymerization of tubulin are potential antitumor agents. For reviews, see E. Hamel, Med. Res. Rev. 16:207-231 (1996) and L. Wang et al., Cancer Chemother Pharmacol, 44:355-361 (1999).
Three major pharmacological sites are present on tubulin: the colchicine site, the vinca alkaloid domain, and the xe2x80x9ctaxoidxe2x80x9d site. The latter site is fully developed only in tubulin polymers with a well-defined protofilament substructure. Parness and Horwitz, J Cell. Biol. 91:479-487 (1981); Takoudju et al., FEBS Lett 227:96-98 (1988).
2. Non-steroidal Modulators of Tubulin Polyimerization.
Colchicine and nocadazole are anti-mitotic drugs that bind tubulin and inhibit tubulin polymerization. When used alone or in combination with other therapeutic drugs, colchicine in particular may be used to treat cancer. See for example PCT application WO 93/03729, and Japanese patent 03240726 (1991). Allocolchicines, with a 7-membered B ring but 6-membered C ring, have been reported, some of which are more active than the corresponding colchicines. Ionio, Heterocycles 22:2207-2211 (1984); Kang et al. J Biol. Chem. 265:10255-10259(1990)).
There are several cytotoxic vinca alkaloids that operate by the mechanism of inhibition of tubulin polymerization. P. Verdier-Pinard et al., Biochem. Pharmacol. 58:959-971 (1999). In particular, the vinca alkaloid vinorelbine (NAVELBINE(trademark)) is a potent inhibitor of tubulin polymerization that is currently approved for certain solid tumors. (Piccart, Cancer Treat. Rev., 23:S59 (1997). Cryptophycin is an even more potent inhibitor that is currently under investigation. D. Panda, Biochemistry, 36:12948 (1997).
Paclitaxel (TAXOL(trademark)) and docetaxel (TAXOTERE(trademark)) are examples of the taxane class of antimitotics, which bind to microtubules much more strongly than they do to individual tubulin molecules. They have the effect of accelerating tubulin polymerization, and stabilizing the microtubules against disassembly, which prevents successful completion of the mitotic process. For reviews, see Rowinsky, Ann. Rev. Med., 48:353 (1997), deFurla, Phytomedicine 4:273 (1997), and Balasubramanian et al., Ann. Reports Med. Chem., 33:151-162. These compounds are moderately effective against certain solid tumors. A combination of paclitaxel with vinorelbine has recently been approved by the F.D.A.
Compounds with similar biological effects to those of paclitaxel and docetaxel include discodermolide, eleutherobin, sarcodictyin, and the epothilones. These compounds are in various stages of study and/or development as anti-tumor agents.
3. Steroidal Modulators of Tubulin Polymerization.
Among antimitotic agents that appear to bind at the colchicine site are synthetic analogs of estradiol, such as diethylstilbestrol and estramustine, and the major endogenous metabolite of estradiol, 2-methoxyestradiol (xe2x80x9c2MExe2x80x9d). See for example R. D""Amato et al., Proc Natl Acad Sci USA, 91:3964-3968 (1994); M. Lottering et al., Cancer Res. 52:5926-5923 (1992); L. Spicer and J. Hammond, Mol. and Cell. Endo. 64:119-126 (1989); S. Rao and Engelberg, J Exp. Cell Res. 48:71-81 (1967).
2-Methoxyestradiol (2ME) is a naturally occurring mammalian metabolite of estradiol; it has very low affinity for the estrogen receptor. H. Breuer and R. Knuppen, Naturwissenschafte 12:280-281 (1960); H. Gelbke and R. Knuppen, Steroid Biochem., 7:457-463 (1976). Interest in 2ME has been stimulated by its cytotoxicity in cancer cell cultures, which is characterized by uneven chromosome distribution, faulty spindle formation, inhibition of DNA synthesis and mitosis, and an increase in the number of abnormal metaphases. J. Seegers et al., J Steroid Biochem. 32:797-809 (1989); M. Cushman et al,. J Med. Chem. 38:2041-2049 (1995).
2ME has been shown to bind to the colchicine binding site of tubulin, resulting in inhibition of tubulin polymerization and/or formation of polymer with altered stability properties and morphology. Hamel et al., Biochemistry 35:1304-1310 (1996). Recent in vitro and in vivo results have shown that 2ME inhibits angiogenesis and tumor growth. Fotsis et al., Nature 368:237-239 (1994); Klauber et al., Cancer Res. 57:81-86 (1997).
Efforts have been made to investigate the structure-activity relationships of 2ME and its analogues, in an effort to design more potent anticancer agents. Most work has been directed at modifications in the steroid A ring, which is presumed to be analogous to the colchicine tropolonic C ring. Among the compounds reported, 2-ethoxyestradiol (2EE) has greater inhibitory effects on tubulin polymerization and is 10-fold more cytotoxic than 2ME. Some 6-substituted 2-ethoxyestradiols were synthesized and also demonstrated promising biological activities. Estradiol analogs bearing acetyl groups at positions C-2 and/or C-17 have also been evaluated for their effects on tubulin polymerization, but had minimal or no effect on tubulin polymerization. H.-M. He and M. Cushman, Bioorg. Med. Chem. Lett. 4:1725-1728 1994); M. Cushman et al., J. Med. Chem. 38:2041-2049 (1995); M. Cushman et al., J. Med. Chem. 40:2323-2334 (1997).
Miller et al., J Med. Chem. 40:3836-3841 (1997) disclosed 7-membered tropolonic A ring analogs of 2ME, which were designed to enhance the similarity of the steroid A ring to the C ring of colchicine. They found several of these A-homoestranes to be highly active inhibitors of tubulin assembly.
4. B-Homoestra-1 .3.5(10)-trienes.
B-Homoestra-1,3,5(10)-trienes are a little-known and very little-studied class of compounds. There have been few reported syntheses of such compounds, and even fewer biochemical or pharmaceutical studies. To the best of the present inventors"" knowledge, the five references discussed below constitute the known prior art in this area.
L. W. Rampy, in a thesis entitled xe2x80x9cTotal Synthesis of B-homoestrone and Approaches to Azaestronesxe2x80x9d (1967, University of Michigan, Ann Arbor Mich.; Chemical Abstracts 68:96028; Diss. Abstr. B 1967, 28(6):2364) described the first total synthesis of racemic xe2x80x9cB-homoestronexe2x80x9d (3-hydroxy-B-homoestra-1,3,5(10)-trien-17-one). Eleven intermediates and derivatives having 3-hydroxy and 3-methoxy groups were described. None of the compounds disclosed had more than a single hydroxy or methoxy group on the A ring, and this group was always at the 3-position. No biological or biochemical activity of the compounds was disclosed, although xe2x80x9cB-homoestronexe2x80x9d was prepared for the stated purpose of investigating its potential as a modulator of plasma lipid levels.
E. E. Galantay, in French patent 1548354 (1968), disclosed, inter alia, the synthesis in racemic form of B-homoestra-1,3,5(10),8,14-pentaenes, having hydroxy, alkoxy, and acyloxy substituents at position 3. None of the disclosed compounds had more than a single substituent on the A ring, and the substituent was always at the 3-position. The compounds were claimed to be useful for modulating estrogen-dependent conditions, e.g., endometriosis, dysmenorrhea, osteoporosis, and atherosclerosis, but no utility for the treatment of cancer was suggested.
E. E. Galantay and H. P. Weber, Experientia (1969) 25(6):571-572, described the total synthesis of racemic xe2x80x9cB-homoestronexe2x80x9d and some derivatives, referring to them as xe2x80x9ca hitherto unknown class of compounds.xe2x80x9d These authors were evidently unaware of the previous synthesis of B-homoestrone by L. Rampy. An X-ray crystal structure of the 3-methoxy derivative was reported. None of the compounds disclosed had more than a single methoxy or hydroxy group on the A ring, and this substituent was always at the 3-position. No biological or biochemical activity of the compounds was disclosed.
E. Velarde, L. H. Knox, A. J. Cross, and P. Crabbe, Justus Liebigs Ann. Chem. (1971) 748:123-133, described 7-fluoro-B-homoestra-1,3,5(10)-trienes, having hydroxy, methoxy, and acetoxy substituents at position 3. None of the disclosed compounds had more than a single substituent in the A ring, and all were at the 3-position. No biological or biochemical activity of the compounds was disclosed.
M. B. Groen and F. J. Zeelen, Recl.: J. R. Neth. Chem. Soc. (1984) 103(5):169-173, described the preparation of 1-methoxy and 3-methoxy B-homo-gona-1,3,5(10)-triene steroids, and subsequent transformations of the D ring to provide 3-methoxy-B-homoestra-1,3,5(10)-trien-17-one. However, none of the compounds described by Groen and Zelen had more than a single methoxy group on the A ring. No biochemical, pharmacological, or biological activity of the compounds was disclosed.
In addition to the synthesis and characterization of B-homoestra-1,3,5(10)-trienes described in the above references, these compounds fall into one of the many large classes of compounds generically represented, but neither prepared nor characterized, in U.S. Pat. Nos. 5,504,074 and 5,661,143. These patents are incorporated herein by reference in their entireties.
5. Summary
Although paclitaxel and docetaxel in particular have proved to be medical and commercial successes, all of the active tubulin- or microtubule-modulating agents described above suffer from toxicity problems, such as bone marrow suppression, hair loss, diarrhea, etc. They also tend to be specific for a limited class of tumor types. There remains a need for newer modulators of tubulin polymerization which may prove to have improved selectivity and reduced side-effects.
1. Definitions.
xe2x80x9cDiseases characterized by undesirable cell mitosisxe2x80x9d includes but is not limited to excessive or abnormal proliferation of endothelial cells (e.g., psoriasis, atherosclerosis, endometriosis, hyperplasias), solid tumors and tumor metastasis, benign tumors, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, vascular malfunctions, abnormal wound healing, inflammatory and immune disorders, Bechet""s disease, gout or gouty arthritis.
xe2x80x9cDiseases characterized by undesirable angiogenesisxe2x80x9d includes but is not limited to hematomas, and angiogenesis accompanying: solid tumors, rheumatoid arthritis, psoriasis, diabetic retinopathy and other ocular angiogenic diseases such as retinopathy of prematurity (retrolental fibroplasic), macular degeneration, corneal graft rejection, neovascular glaucoma and Osler Weber syndrome. Normal but undesired processes involving angiogenesis, such as menstruation and the implantation of a blastula, are intended to fall within the meaning of the phrase as well.
The terms xe2x80x9calkylxe2x80x9d and xe2x80x9cacylxe2x80x9d as used herein are intended to include both straight-chain and branched alkyl and acyl groups.
xe2x80x9cMAP(s)xe2x80x9d refers to microtubule-associated protein(s), as described in E. Hamel and C. Lin, Biochemistry, 23:4173-4184 (1984).
2. Description of the Invention
It has been discovered that certain compounds within the scope of the claims below modulate tubulin polymerization and microtubule formation and/or depolymerization, and therefore are expected to be useful for treating mammalian diseases characterized by undesired cell mitosis and/or undesired angiogenesis.
One object of this invention is to provide B-ring expanded estra-1,3-5(10)-triene derivatives of formula 1: 
wherein the groups R1, R2, R3, A and B are as described further hereinbelow.
Another object of the invention is to provide pharmaceutical compositions comprising the compounds of the invention, and methods of treating mammals in need of anti-tumor or anti-angiogenic therapy with these compositions.
Yet another object of the invention is to provide methods of making 2,3-disubstituted B-homoestra-1,3,5(10)-trienes of formula 1.